Tuberculosis (TB) remains a major cause of morbidity and mortality worldwide, particularly in much of the developing world. In regions where HIV and TB are co-prevalent, TB is a leading cause death in HIV-infected individuals. Where MDR-TB has emerged, including in the U.S., treatment is extremely difficult, mortality rates are high, and persons with HIV/AIDS have been disproportionately affected. New approaches to prevention and treatment, including new drugs that act through novel targets, are needed to treat drug- susceptible and MDR-TB more effectively. A signal transduction pathway mediated by the eukaryotic-like transmembrane serine/threonine kinases PknA and PknB is essential for the viability of M. tuberculosis. Data from the Pl's laboratory and the M. tuberculosis genome sequence indicate that this signaling pathway regulates cell wall synthesis and cell division. The underlying hypothesis of this R21 proposal is that the extracellular domain of PknB (Ed-PknB) functions in vivo as a receptor domain or as a localization domain, through interaction with extracellular molecule(s), and that these interactions are essential for the function of PknB. The goals of this research are to gain insight into the PknA/B signaling network through identification and characterization of synthetic ligands of ED-PknB, and to determine whether such ligands have potential as leads for novel anti-tuberculars. These goals will be addressed by the following specific aims and methods: 1) screening small molecule chemical libraries for compounds that bind to ED-PknB using a novel glass slide microarray technology, validating positives and quantifying the strength of interactions by surface plasmon resonance, and 2) investigating the effects of the interaction of these ligands, and active derivates, on mycobacterial viability and cell morphology, and on PknB kinase activity. The long-term goals of this research are to gain insight into the PknA/B signaling pathways, and to determine whether synthetic ligands of ED-PknB have potential as leads for new approaches to the treatment or prevention of TB.